1. Field of the Invention
The present invention refers to compounds with antiviral activity and, more particularly to conjugated compounds of antiviral drug with carriers having hepatotropic activity.
2. Description of the Related Art
In the treatment of the infection caused by viruses, the side effects produced by antiviral drugs can be reduced with the adoption of the chemotherapeutic lysosomotropic approach (Balboni P G, Minia A, Grossi M P, Barbanti-Brodano G, Mattioli A, Fiume L., Activity of albumin conjugates of 5-fluorodeoxyuridine and cytosine arabinoside on poxviruses as a lysosomotropic approach to antiviral chemotherapy, Nature 1976; 264: 181-183).
This consists in conjugating the drug to a macromolecule that is selectively captured from the infected cells and transported in the lysosomes therefrom.
If, as desired, the lysosomal enzymes break the linkage between the carrier and the drug, the latter results as being concentrated in a pharmacological active form within the infected cells. EQU AZTMP=3-azido-3-deoxythymidine EQU ACV=acyclovir
The chronic hepatitis by B (HBV) virus and by C (HCV) virus are a proper target for this chemotherapeutic approach because: (a) these viruses grow especially in the hepatocytes; (b) hepatocytes specifically bring inside and transport in the lysosomes some glycoproteins with galactose residues that can therefore function as hepatotropic vectors of drugs; (c) the conjugates of drug/glycoproteins can easily come into contact with the surface of the hepatocites inasmuch the hepatic sinusoid are not a barrier for proteins.
Following this approach and to reduce its neurotoxic side effects, the arabinoside adenine monophosphate antiviral drug (ara-AMP), active against HBV (Jacyna M R, Thomas H C., Antiviral therapy: Hepatitis B., Brit Med Bull 1990; 46: 369-382), has been conjugated with asialophetuin (AF) (Fiume L; Mattioli A, Busi C, Balboni P G, Barbanti-Brodano G, De Vries J, Altman R, Wieland Th., Selective inhibition of Ectromelia virus DNA synthesis in hepatocytes by adenine-9-.beta.-D-arabinofuranoside (ara-A) and adenine-.beta.-D-arabinofuranoside 5'-monophosphate (ara-AMP) conjugated to asialofetuin, FEBS LEtters 1980; 116: 185-188) and with the lactosaminated albumin (L-SA) (Fiume L, Busi C, Mattioli A, Balboni P G, Barbanti-Brodano G., Hepatocyte targeting of adenine-9-.beta.-D-arabinofuranoside 5'-monophosphate (ara-AMP) coupled to lactosaminated albumin, FEBS Lett 1981; 129: 261-264; Fiume L, Bassi B, Busi C, Mattioli A, Spinosa G., Drug targeting in antiviral chemotherapy. A chemically stable conjugate of 9-.beta.-D-arabinofuranosyladenine 5'-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells, Biochem Pharmacol 1986; 35: 967-972). In mice, both these two carriers have brought about a hepatic targeting of the drug.
The L-SA has a great advantage on AF: infact, the conjugates prepared with homologous lactosaminated albumin (that is, of the same species), when introduced intravenously, do not induce the antibodies formation (Fiume L, Mattioli A, Busi C, Spinosa G, Wieland Th., Conjugates of adenine-9-.beta.-D-arabinofuranoside monophoshate (ara-AMP) with lactosaminated homologous albumin are not immunogenic in the mouse, Experientia 1982; 38: 1087-1089; Fiume L., Busi C., Preti P., Spinosa G. Coniugates of ara-AMP with lactosaminated albumin: a study on their immunogeneticity in mouse and rat. Cancer Drug Delivery 1987; 4: 145-150). In woodchuck with hepatitis by WHV (Ponzetto A, Fiume L, Forzani B, Song S Y, Busi C, Mattioli A, Spinelli C, Marinelli M, Smedile A, Chiaberge E, Bonino F, Gervasi G B, Rapicetta M, Verme G., Adenine arabinoside monophosphate and acyclovir monophosphate coupled to lactosaminated albumin reduce woodchuck hepatitis virus viremia at doses lower than do the unconjugated drugs, Hepatology 1991; 14: 16-24) and in patients with chronic infection by HBV, (Fiume L., Torrani Cerenzia M R, Bonino F, Busi C, Mattioli A, Brunetto M R, Chiaberge E Verme G. Inhibition of hepatitis B virus replication by vidarabine monophosphate conjugated with lactosaminated serum albumin, Lancet 1988; 2: 13-15. Torrani Cerenzia M R, Fiume L, Busi C, Mattioli A, Di Stefano G, Gervasi G B, Brunetto M R, Piantino P. Verme G, Bonino F. Inhibition of hepatitis B virus replication by adenine arabinoside monophosphate coupled to lactosaminated albumin. Efficacy, minimal effective dose and plasma clearance of conjugate. J Hepatol--1994; 20: 307-309), the ara-AMP conjugated to the L-SA has inhibited the viral replication at doses 3-6 times lower than those of the free drug.
The conjugate with L-SA should be administered intravenously owing to the high volume needed for the injection and because, by other ways antibodies are produced, and this results in a not good patient compliance in long-lasting treatments.
A hepatotropic carrier of ara-AMP and of other antiviral drugs allowing the intramuscular administration of the corresponding conjugates would therefore be a remarkable improvement from the therapeutic point of view.
It is also known that a basic polyaminoacid, the poly-L-lysine, with one third of the amino-groups substituted with galactose residues, if intravenously administered, performs a hepatic targeting of ara-AMP (Fiume L, Bassi B, Busi C, Mattioli A, Spinosa G, Faulstich H., Galactosylated poly(L-lysine) as a hepatotropic carrier of 9-.beta.-D-arabinofuranosyladenine 5'-monophosphate, FEBS Letters 1986; 203: 203-206). Moreover, the poly-L-lysine, when all or large part of its .epsilon.-amino groups are substituted, does not form antibodies even when administered by ways different from the intravenous injection (Levine B B, Studies on antigenicity. The effect of succinylation of .epsilon.-amino groups on antigenicity of benzylpenicilloyl-poli-L-lysine conjugates in random-bred and in strain 2 Guinea pig, Proc Soc Exptl Biol Med 1964; 116: 1127-1131; Sela M. Immunological studies with synthetic polypeptides, Advan Immunol, 1966; 5: 29-129).
It has now been found that if in the poly-L-lysine most of the .epsilon.-amino groups are substituted with the galactose and with one of the antiviral drugs known for performing their activity against hepatic viruses, three very important therapeutic effects are produced in mice:
(i) the conjugate loses the poly-L-lysine high toxicity which distinguished the previous galactosylated poly-L-lysine-ara-AMP conjugates (Fiume et al, FEBS Letters 1986, 203, 203-206) in which most the .epsilon.-amino groups remained unsubstituted; PA1 (ii) the antiviral drug hepatic targeting is brought on even if the conjugate is not administered intravenously, and particularly, by intramuscular injection. PA1 (iii) the repeated conjugate administration by intramuscular and intravenous injection does not produce antibodies. PA1 (a) conjugation of the basic polyaminoacid with antiviral drug or with galactose residues and PA1 (b) subsequent conjugation of the conjugate resulting from step (a) with galactose residues or antiviral drugs residues, respectively. In the previous general definition it can be noticed that the two conjugation steps may be inverted.
The therapeutic importance of this property will be evident if it is considered that the possibility to achieve the hepatic targeting by intramuscular injection not only involves to utilize its interesting peculiar characteristics already previously mentioned in relation to the lysosomotropic approach (i.e. drastic reduction of the side effects relevant to the toxicity of the antiviral drugs), but also as much as important to make much more comfortable for the patient to undergo the chemotherapeutic-treatment which, as a rule in the viral chronic hepatitis, is long lasting.
In the preferred definition, the basic poliaminoacid is selected between poly-L-lysine and poly-L-ornithine and the drugs among those known for their activity against the hepatic viruses, and particularly among ara-AMP, acyclovir, ribavirin, azidothymidine, and the like.
The present invention refers therefore to the use, as carrier of antiviral drugs, of a basic, galactosylated polyaminoacid, said polyaminoacid being characterized in that most of the amino groups are substituted with molecules of the drug and with galactose molecules.
This high degree of substitution eliminates the acute toxicity of both the basic polyaminoacids and the conjugates of poly-L-lysine previously published (Fiume et al. FEBS Letters 1986; 203: 203-206) in which less than 50% of .epsilon.-amino groups were substituted by the drug and by the galactosyl residues.
The preparation of the conjugated compounds according to the present invention provides a two-steps procedure:
As a rule, the choice is suggested by the polyaminoacid molecular weight in a sense that, with lower molecular weights, it is preferred to carry out first the conjugation with the drug and the conjugation with galactose thereafter.